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Flow cytometer calibration method

  • US 5,837,547 A
  • Filed: 12/27/1995
  • Issued: 11/17/1998
  • Est. Priority Date: 12/27/1995
  • Status: Expired due to Term
First Claim
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1. A method for increasing accuracy, precision and reliability of studies of protein-labeled cell samples using a flow cytometer or fluorescent microscope, comprising:

  • (a) providing a microbead suspension containing;

    (i) a plurality of specific populations of microbeads which bind and are in equilibrium with a saturating amount of a selected protein; and

    (ii) a microbead population having no binding capacity for said selected protein;

    (b) providing information on calibrated binding capacities of the microbead populations;

    (c) using the microbead suspension to obtain a calibration plot by a method selected from the group consisting of;

    (i) washing the microbead suspension, analyzing the washed suspension on the flow cytometer or fluorescence microscope to determine representative peak channels, and using the representative peak channels and the calibrated binding capacities to obtain a calibration plot for the flow cytometer or fluorescence microscope, to obtain performance parameters and determine protein binding of cells; and

    (ii) adding the microbead suspension without washing, to sample cells, which fluorescently labels the sample cells due to the excess labeled protein, to form a mixture of microbeads and sample cells, incubating the mixture, washing the mixture, analyzing the washed mixture by gating on the microbeads in the mixture using the flow cytometer or fluorescence microscope to determine representative peak channels, and using the representative peak channels and the calibrated binding capacities to obtain a calibration plot for the flow cytometer or fluorescence microscope, to obtain performance parameters for using the flow cytometer or fluorescence microscope;

    wherein gating on the fluorescently-labeled sample cells with the flow cytometer or fluorescence microscope allows determination of the antibody-binding capacity of the sample cells.

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